The tripeptide thiol glutathione (L-.gamma.-glutamyl-L-cysteinylglycine; GSH) is found within virtually all cells. It functions in metabolism, transport, and cellular protection. Specifically, for example, glutathione participates in transhydrogenation reactions that are involved in the formation and maintenance of the sulfhydryl group of other molecules (e.g., coenzyme A, various enzymes, and other proteins). Glutathione provides reducing capacity for various reactions, e.g., the formation of deoxyribonucleotides by ribonucleotide reductase. Glutathione also functions in the detoxification of hydrogen peroxide, other peroxides, and free radicals. In addition, glutathione plays a role in detoxification of a variety of foreign compounds which interact with glutathione and which are ultimately excreted in the form of mercapturic acids. Analogous derivatives of glutathione are formed with endogenous metabolites, e.g., in the metabolism of leukotrienes, prostaglandins, steroids, and melanins. There is also evidence that the .gamma.-glutamyl moiety of glutathione functions in the transport of amino acids (especially cysteine and certain neutral amino acids) and possibly also of peptides and amines.
Glutathione synthesis takes place within almost all animal cells and in those of many plants and microorganisms. The two enzymes required for the synthesis of this tripeptide (.gamma.-glutamylcysteine synthetase and glutathione synthetase) have been isolated from a number of different sources (Dolphin, D., Poulson, R. and Avramovic, O. (eds.) (1989), Glutathione: Chemical, Biochemical, and Medical Aspects, Parts A and B, Coenzyme and Cofactors Series, Vol. III John Wiley, New York; Snoke, J. E. and Bloch, K. (1952), J. Biol. Chem. 199, 407-414; Snoke, J. E. (1955), J. Biol. Chem. 213, 813-842; Meister, A. (1974), in The Enzymes (Boyer, P. D., ed) 3rd Ed, Vol. 10, pp. 671-691, Academic Press, N.Y.).
Gamma-glutamylcysteine synthetase catalyzes the first and rate-limiting step of GSH synthesis (reaction (1)): ##STR1## Gamma-glutamylcysteine is feedback inhibited by GSH. Richman, P., and Meister, A. (1975) J. Biol. Chem. 250, 1422-1426; Huang, C.-S., Chang, L.-S., Anderson, M. E., and Meister, A. (1993) J. Biol. Chem. 268, 19675-19678).
The amino acid sequences of the two separately coded proteins that comprise the two subunits of .gamma.-glutamylcysteine synthetase in mammalian tissues have been deduced. Yan, N., and Meister, A. (1990), J. Biol. Chem. 265, 1588-1593; Huang, C.-S., Anderson, M. E., and Meister, A. (1993), J. Biol. Chem. 268, 20578-20583. This enzyme, which differs substantially in subunit structure and amino acid sequence from bacterial .gamma.-glutamylcysteine synthetase, has been the subject of several studies. Yan, N., and Meister, A. (1990), J. Biol. Chem. 265, 1588-1593; Huang, C.-S., Anderson, M. E., and Meister, A. (1993), J. Biol. Chem. 268, 20578-20583; Huang, C.-S., Moore, W., and Meister, A. (1988), Proc. Natl Acad. Sci., U.S.A. 85, 2464-2468; Seelig, G. F., and Meister, A. (1985), Methods in Enzymology 113, Chapter 47, pp. 379-390; Seelig, G. F., and Meister, A. (1984), J. Biol. Chem. 259, 3534-3538.
Glutathione synthetase, which catalyzes the synthesis of GSH from .gamma.-glutamylcysteine and glycine, (reaction (2)): ##STR2## has also been purified from several biological sources. Meister, A. (1974), in The Enzymes (Boyer, P. D., ed) 3rd Ed, Vol. 10 pp. 671-691, Academic Press, N.Y. The GSH synthetase of E. coli was isolated as described in Gushima, H., Miya, T., Murata, K., and Kimura, A. (1983), J. Appl. Biochem. 5, 210-218 and Gushima, H., Yasuda, S., Soeda, E., Yokota, M., Kondo, M., and Kimura, A. (1984), Nucl. Acids Res. 12, 9299-9307. The purified E. coli enzyme, which had a M.sub.r 38,000, was cloned and sequenced. The enzymes purified from baker's yeast, described in Mooz, E. D. and Meister, A. (1967), Biochemistry, 6, 1722-1734, and fission yeast, described in Mutoh, N., Nakagawa, C. W., Ando, S., Tanabe, K., and Hayashi, Y. (1991), Biochem. Biophys. Res. Comm. 181, 430-436, each have a molecular weight of about 120,000. The fission yeast enzyme is reported to be a heterotetramer composed of two subunits with molecular weights of 33,000 and 26,000, respectively. See Mutoh, N., Nakagawa, C. W., Ando, S., Tanabe, K., and Hayashi, Y. (1991), Biochem. Biophys. Res. Comm. 181, 430-436. The DNA that codes for the heavy subunit was isolated and partially sequenced. When the heavy subunit DNA was introduced into the yeast, both the heavy subunit and the light subunit were overexpressed. The authors concluded that the enzyme is composed of 2 kinds of subunits and that it has an A.sub.2 B.sub.2 structure. The gene for the large subunit of Schizosaccharomyces pombe was cloned from a S. pombe genomic DNA library by complementation of cadmium ion hypersensitivity of a GSH synthetase deficient S. pombe mutant, as described in Hayashi, Y., Nakagawa, C. W. and Mutoh, N. (1991), Biochem. Cell Biol., 69, 115-121. Cadmium ions (and certain other metal ions) induce formation of phytochelatins, which have the general structure: (.gamma.-glu-cys).sub.n -gly. See Grill, E., Loffler, S., Winnacker, E-L, and Zenk, M. H. (1989), Proc. Natl. Acad. Sci. USA, 86, 6838-6842; (cadystins, Hayashi, Y., Nakagawa, C. W. and Mutoh, N. (1991) Biochem. Cell Biol., 69, 115-121). The mechanisms involved in the formation of these peptides are still under study, and the possibility that GSH synthetase activity is involved has been suggested. See Hayashi, Y., Nakagawa, C. W. and Mutoh, N. (1991) Biochem. Cell Biol., 69, 115-121. Putative cDNA for frog GSH synthetase was isolated by using degenerative oligonucleotides derived arbitrarily from the deduced fission yeast amino acid sequence. Habenicht, A., Hille, S., and Knochel, W. (1993), Biochem Biophys. Acta 1174, 295-298. The proposed subunit structure of the frog enzyme needs to be substantiated since the enzyme has not yet been isolated. However, expression of the enzyme using the putative frog cDNA has not been reported, and no activity for an enzyme encoded by the frog cDNA is known.
Rat kidney glutathione synthetase has also been isolated. Oppenheimer, L., Wellner, V., Griffith, O., and Meister, A. (1979), "Glutathione Synthetase Purification From Rat Kidney and Mapping of the Substrate Binding Sites," J. Biol. Chem., 254, 5184-5190. However, the primary structure of the mammalian enzyme has not previously been described. Although, as noted above, some data on the amino acid sequences of the GSH synthetase of lower forms (e.g., bacteria, yeast) are available, there are major differences between certain properties of these enzymes and those of the rat kidney enzyme, for example, molecular weight, subunit structure, and inhibition by iodoacetamide.
Glutathione synthetase deficiency in humans is associated with potentially serious health complications. Two general types of such deficiency have been observed. Meister, A., and Larsson, A. (1994), "Glutathione Synthetase Deficiency and Other Disorders of the .gamma.-Glutamyl Cycle," in The Metabolic Basis of Inherited Disease, (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D. eds.) 7th Ed., in press. In one, an unstable form of GSH synthetase is expressed, leading to an apparently selective deficiency of GSH in the erythrocyte. In 5-oxoprolinuria, another type of GSH synthetase deficiency, dramatic and potentially fatal metabolic consequences occur as a result of over-production of 5-oxoproline which leads to severe metabolic acidosis. In this condition, there is over-production of .gamma.-glutamylcysteine, whose synthesis is not feedback inhibited because of the low levels of GSH and possibly because there is induction of .gamma.-glutamylcysteine synthetase. .gamma.-Glutamylcysteine is converted by the action of .gamma.-glutamylcyclotransferase to cysteine and 5-oxoproline. Cysteine is used by .gamma.-glutamylcysteine synthetase (in a futile cycle), and 5-oxoproline accumulates in amounts that exceed the capacity of 5-oxoprolinase to convert it to glutamate. This leads to substantial accumulation of 5-oxoproline and to its urinary excretion in amounts that may be as high as 30 grams per day (normally &lt;0.14 g. per day). Meister, A., and Larsson, A. (1994), "Glutathione Synthetase Deficiency and Other Disorders of the .gamma.-Glutamyl Cycle," in The Metabolic Basis of Inherited Disease, (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D. eds.) 7th Ed., in press. Severe damage to the central nervous system, and even potentially death, are among the potential complications.
Modifications of glutathione metabolism are sometimes desirable even in persons having normal glutathione levels. Such modifications may be achieved by administration of selective enzyme inhibitors to decrease intracellular glutathione levels, or by providing compounds that increase glutathione synthesis. Such effects are useful in chemotherapy and radiation therapy and in protecting cells against the toxic effects of drugs, other foreign compounds and oxygen.
Modification of GSH metabolism to deplete or increase cellular GSH may serve various purposes. For instance, it has long been known that thiols protect cells against the effects of irradiation. Since decreasing cellular GSH makes cells more susceptible to irradiation, glutathione depletion is useful in chemotherapeutic situations in which the cells to be killed and the cells to be spared have substantially different quantitative requirements for GSH. Depletion of GSH by inhibition of its synthesis also serves as a valuable adjuvant in chemotherapy with drugs that are detoxified by reactions involving GSH.
Conversely, development of resistance to a drug or to radiation may be associated with an increase in cellular GSH. GSH serves effectively in the detoxification of many drugs, and it is known that a significant pathway of acetaminophen detoxification involves conjugation with GSH.
Treatment with a thiazolidine such as L-2-oxothiazolidine-4-carboxylic acid, may be of value to patients with liver disease and to premature infants who may be deficient in the utilization of methionine sulfur for cysteine formation, and thus in GSH synthesis. The effectiveness of such a thiazolidine as an intracellular cysteine precursor depends on the presence of 5-oxoprolinase, an enzyme activity found in almost all animal cells.
Various methods are known to increase cellular levels of glutathione. Glutathione is composed of three amino acids: glutamic acid, cysteine and glycine. Administration to animals of the amino acid precursors of
glutathione may produce an increase in cellular glutathione, but there is a limit to the effectiveness of this procedure. Concentrations of GSH are dependent on the supply of cysteine, which is derived from dietary protein and by trans-sulfuration from methionine in the liver. Administration of cysteine is not an ideal way to increase GSH concentration because cysteine is rapidly metabolized and furthermore, it is very toxic. Administration to animals of compounds that are transported into cells and converted intracellularly into cysteine is sometimes useful in increasing cellular glutathione levels. For example, the thiazolidine L-2-oxothiazolidine-4-carboxylate is transported into the cell, where it is converted by 5-oxoprolinase to L-cysteine, which is rapidly used for GSH synthesis.
Another way in which tissue GSH concentration may be increased is by administration of .gamma.-glutamylcysteine or of .gamma.-glutamylcysteine, as described in U.S. Pat. No. 4,879,370, which is hereby incorporated by reference. The administered .gamma.-glutamyl amino acid is transported intact and serves as a substrate of GSH synthetase. It is also known that administration of N-acetyl-L-cysteine increases tissue concentrations of GSH, and although glutathione itself is not effectively transported into cells, half-esters and di-esters (ethyl, isopropyl, etc.) of glutathione are transported into, for example, liver and kidney cells, and so increase cellular glutathione levels, also as described in U.S. Pat. No. 4,879,370. However, complications limit the usefulness of these methods. For example, successful administration of N-acetyl-L-cysteine depends on the presence of de-acetylase (Greenstein, J. P. and Winitz, M. Chemistry of the Amino Acid, Wiley,N.Y. 1960, hereby incorporated by reference), and both .gamma.-glutamylcysteine synthetase and glutathione synthetase, and ATP. Transporters for dipeptides, which apparently are not present in every cell, are required for treatment by administration of .gamma.-glutamylcysteine or .gamma.-glutamylcystine, which further requires glutathione synthetase and ATP. Glutathione esters are effective, but the effects are not long lasting, as the esters are metabolized relatively rapidly.
The present invention is directed toward overcoming these deficiencies.